The melanocortin 4 receptor (MC4R) has been shown to be an important regulator of energy homeostasis in both rodents and humans by decreasing food intake and increasing energy expenditure. Targeted deletion of MC4R in rodents resulted in a marked increase in food intake or hyperphagia, reduced energy expenditure, and obesity. Although MC4R expression is restricted primarily to the brain, it is found in multiple sites within the central nervous system. It is currently unknown if MC4Rs in one of these sites is responsible for regulating energy homeostasis or if it is distributed through several sites. Previous work in our laboratory demonstrated that when MC4R expression is restored on neurons of the PVN and a small subset of neurons on the amygdala in MC4R null mice, 60% of the obesity was prevented and the hyperpahgia was completely rescued. However, no effect on the decreased energy expenditure was observed. These findings therefore established that MC4Rs on neurons of the PVN/amygdala were important for the normal control of food intake while MC4Rs on other neuronal sites might not play a role. However, recent studies suggest that MC4Rs on neurons in the hindbrain mediate similar effects on food intake. This would then suggest that although MC4Rs on PVN/amygdala neurons are important, there is a functional redundancy in the MC4R pathways which regulate food intake. This issue must be resolved if we are to understand how the MC4Rs regulate food intake. Therefore, the overall goal of the studies proposed below is to determine where the relevant MC4Rs that control food intake are located. We hypothesize that 1) MC4Rs on PVN/amygdala neurons are the only site in the brain where MC4Rs mediate its effect on food intake and 2) the relevant MC4Rs are found on the presynaptic axon terminals of the distant projections of PVN/amygdala neurons. Specific Aim 1: To determine if PVN/amygdala neurons are the only site in the brain where MC4Rs control food intake. Specific Aim 2: To determine where the relevant MC4Rs on PVN/amygdala neurons that regulate food intake are located. Specific Aim 3: To determine if MC4Rs on GABAergic neurons mediate effects on food intake. The MC4R pathways are critical in the regulation of body weight as a malfunction in this system results in obesity. Identification of the sites in the brain by which MC4Rs regulate body weight will allow for the development of more effective therapeutics to treat and prevent obesity.